HGH, Page 1
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Phone: (703) 266-5667, FAX: (703) 266-5664
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Enzyme Immunoassay for the
Quantitative Determination of
Human Growth Hormone
(HGH) Concentration in
Human Serum
I. Introduction
II. Principle of the test
III. Intended use
IV. Materials and components
V. Specimen collection and
preparation
VI. Storage of test kit and
instrumentation
VII.Reagent preparation
VIIIAssay procedures
IX. Calculation of results
X. Example of standard curve
XI. Expected values and sensitivity
XII.References
I.
Introduction
Human growth hormone (HGH, somatotropin) is a
polypeptide secreted by the anterior pituitary. It is
191 amino acids in length and has a molecular mass
of approximately 22,000 daltons. Its metabolic
effects are primarity anabolic. HGH promotes
protein conservation and is engaged in a wide range
of mechanisms for protein synthesis. It also
enhances glucose transport and facilitates glycogen
storage. Its cascade of growth-promoting action is
mediated by another family of peptide hormones,
the somatomedins. HGH measurement is primarily
of interest in the diagnosis and treatment of various
forms of abnormal growth hormone secretion.
Disorders caused by hyposecretion include
dwarfism and unattained growth potential, and
hypersecretion is associated with gigantism and
acromegaly.
HUMAN GROWTH HORMONE
(HGH) ENZYME
IMMUNOASSAY TEST KIT
Catalog Number:
1901
Caution must be exercised in the clinical
interpretation of growth hormone levels. These
vary throughout the day, making it difficult to
define a normal range or to judge an individual’s
status based on a single determination. Many
factors are known to influencec the rate of growth
hormone secretion, including periods of sleep and
wakefulness, exercise, stress, hypoglycemia,
estrogens, corticosteroids and L-dopa. Because of
its similarity to prolactin and placental lactogen,
earlier growth hormone immunoassays were often
plagued with falsely high values in pregnant and
lactating women.
Because not all acromegalic individuals have
elevated baseline levels of growth hormone,
suppression tests based on glucose loading are of
value in this context. In spite of the induced
hyperglycemia, there is rarely a decrease from
baseline levels in acromegaly.
Growth hormone-deficient individuals have fasting
and resting levels similar to those found in normal
individuals. Various challenge tests have therefore
been devised to differentiate them. For example,
with the onset of deep sleep or after 15 to 20
minutes of vigorous exercise, growth hormone
levels normally rise. Other tests of growth
hormone responsiveness are based on the
administration of L-dopa, arginine and insulin.
Propanolol or estrogen are sometimes given in
conjunction with the primary stimulus to accentuate
the response.
A small number of dwartism cases have been
documented in which both the basal level of HGH
and the response to challenge testing were normal.
Such cases may involve tissue insensitivity to either
growth hormone or the somatomedins, or
immunoreactive but biologically inactive growth
hormone.
The Human Growth Hormone Enzyme
Immunoassay provides a rapid, sensitive and
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
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HGH, Page 2
reliable test. There is no cross-reactivity with HCG,
TSH, LH, HGH and prolactin.
II.Principle of the test
The HGH Quantitative Test Kit is based on the
principle of a solid phase enzyme-linked
immunosorbent assay. The assay system utilizes a
polyclonal anti-HGH antibody for solid phase
(microtiter wells) immobilization and a mouse
monoclonal anti-ferritin antibody in the antibodyenzyme (horseradish peroxidase) conjugate
solution. The test sample is allowed to react
simultaneously with the antibodies, resulting in
HGH molecules being sandwiched between the
solid phase and enzyme-linked antibodies. After a
60 minute incubation at room temperature, the
wells are washed with water to remove unbound
labeled antibodies. A solution of TMB is added
and incubated for 20 minutes, resulting in the
development of a blue color. The color
development is stopped with the addition of 2N
HCl, and the color is changed to yellow and
measured spectrophotometrically at 450 nm. The
concentration of HGH is directly proportional to
the color intensity of the test sample.
III.Intended use
For the quantitative determination of human growth
hormone (HGH) concentration in human serum.
IV.Materials and components






V.Specimen collection and
preparation
Serum should be prepared from a whole blood
specimen obtained by acceptable medical
techniques. This kit is for use with serum samples
without additives only.
VI.Storage of test kits and
instrumentation
Unopened test kits should be stored at 2-8C upon
receipt and the microtiter plate should be kept in a
sealed bag with desiccants to minimize exposure to
damp air. Opened test kits will remain stable until
the expiring date shown, provided it is stored as
prescribed above. A microtiter plate reader with a
bandwidth of 10nm or less and an optical density
range of 0-2 OD or greater at 450nm wavelength is
acceptable for use in absorbance measurement.
VII.Reagent preparation
1.
2.
Materials provided with the test
kits:






Antibody-coated microtiter wells.
Reference standard set, contains 0, 2.5, 7.5, 15,
30, and 60 ng/ml (WHO, 1st IRP, 66/217)
HGH, lyophilized.
Enzyme conjugate reagent, 13 ml.
Color Reagent A, 13 ml.
Color Reagent B, 13 ml.
2N HCl, 10 ml.
Materials required but not provided:


Distilled water.
Glass tubes or flasks to mix Color Reagent A
and Color Reagent B.
Vortex mixer or equivalent.
Absorbent paper or paper towel.
Graph paper.
Microtiter well reader.
3.
All reagent should be brought to room
temperature (18-25C ) before use.
To prepare TMB solution, make an 1:1 mixing
of Color Reagent A with Color Reagent B up
to 1 hour before use. Mix gently to ensure
complete mixing. The prepared TMB
substrate reagent is stable at room temperature
in the dark for up to 3 hours. Discard the
excess after use.
Reconstitute each lyophilized standard with
1.0ml distilled water. Allow the reconstituted
material to stand for at least 20 minutes.
Reconstituted standards should be stored
sealed at 2-8C.
VIII.Assay procedures
Precision pipettes: 0.05, 0.1, 0.2, and 1.0 ml.
Disposable pipette tips.
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Phone: (703) 266-5667, FAX: (703) 266-5664
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HGH, Page 3
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Secure the desired number of coated wells in
the holder.
Dispense 50l of standard, specimens, and
controls into appropriate wells.
Dispense 100l of Enzyme Conjugate Reagent
into each well.
Thoroughly mix for 30 seconds. It is very
important to have complete mixing in this
setup.
Incubate at room temperature (18-25C) for 60
minutes. Prepare TMB solution up to one hour
before use.
Remove the incubation mixture by flicking
plate content into a waste container.
Rinse and flick the microtiter wells 5 times
with running tap or distilled water.
Strike the wells sharply onto absorbent paper
or paper towels to remove all residual water
droplets.
Dispense 200l of TMB solution into each
well. Gently mix for 5 seconds.
Incubate at room temperature in the dark for
20 minutes.
Stop the reaction by adding 50l of 2N HCl to
each well.
Gently mix for 30 seconds. It is important to
make sure that all the blue color changes to
yellow color completely.
Read optical density at 450nm with a
microtiter reader within 30 minutes.
X.Example of standard curve
Results of typical standard run with optical density
reading at 450nm shown in the Y- axis against
HGH concentrations shown in the X-axis. This
standard curve is for the purpose of illustration
only, and should not be used to calculate
unknowns. Each user should obtain his or her own
data and standard curve.
HGH (ng/ml)
0.0
1.0
2.5
7.5
15.0
30.0
3.5
3
2.5
2
OD
1.5
1
0.5
0
0
Important Note:
Absorbance (450nm)
0.052
0.253
0.501
1.158
2.075
3.025
10
20
30
HGH Conc. (ng/ml)
The wash procedure is critical. Insufficient
washing will result in poor precision and falsely
elevated absorbances readings.
IX.Calculation of results
XI.Expected values and
sensitivity
Calculate the mean absorbance value (A450) for
each set of reference standards, specimens, controls
and patient samples. Constructed a standard curve
by plotting the mean absorbance obtained from
each reference standard against its concentration in
ng/ml on graph paper, with absorbance values on
the vertical or Y axis and concentrations on the
horizontal or X axis. Use the mean absorbance
values for each specimen to determine the
corresponding concentration of HGH in ng/ml from
the standard curve.
Each laboratory must establish its own normal
ranges based on patient population. A normal
range for human growth hormone levels is difficult
to define because of the normal physiological
fluctuations in HGH concentration. In most adult
subjects at rest, after an overnight fast, the HGH
level in serum is 7 ng/ml or less. Changes in HGH
levels in response to various stimuli gives a more
accurate assessment of pituitary dysfunction
requires provocative tests, either stimulation or
suppression.
The minimal sensitivity of the test is 0.5 ng/ml.
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com
HGH, Page 4
XII.References.
1.
2.
3.
4.
5.
Van Wyk, J.J. and Underwood, L.E. Growth
Hormone, Somatomedins and Growth
Failure.
Hospital Practice, 13:57; 1978.
Fisher, D.A. Evaluation of Anterior
Pituitary Function in:
Radioimmunoassay Manual. Ed. Nicholas,
A.L. and Nelson,
J.C.P. 3498 Nichols
Institute, 1977.
Goldfine, I.D. Medical Treatment of
Acromegaly. Annual Revieew of
Medicine. 29:407; 1978.
Reichlin, S. et al. Hypothalamic Hormones.
Annual Revieew of Medicine. 27:359; 1976.
Rimoin, D.L. and Horton, W.A. Short
Stature. J. Pediatrics. 92:523; 1978
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
http://www.atlaslink-inc.com, info@atlaslink-inc.com